Expression, structural and functional studies of fasciclin I

Year: 1992

DOI: 10.7907/ykbt-jt41

Fasciclin I is a cell surface glycoprotein thought to be involved in growth cone guidance in the embryonic insect nervous system. It is expressed on the cell surfaces of all
peripheral nervous system (PNS) axons, a subset of central nervous system (CNS) axons and on some nonneuronal cells. Fly embryos bearing mutations eliminating expression of
both fasciclin I and the Abelson tyrosine kinase exhibit a severe phenotype in which many axon pathways fail to form. Fasciclin I mediates homophilic adhesion in transfected tissue culture cells, suggesting that it may affect growth cone guidance through homophilic interactions. To facilitate structure-function studies of fasciclin I, we have generated mammalian (CHO) cell lines expressing fasciclin I at a high level. The expressed fasciclin I
protein was released from the cell surface in a soluble form by phospholipase C treatment. Milligram quantities of soluble expressed fasciclin I were purified on an immunoaffinity column. Large single crystals were obtained that diffracted to ~5 Å resolution which is
insufficient for a structure determination to atomic resolution by x-ray crystallography. In an effort to produce a form of fasciclin I more amenable to crystallization, we also generated CHO and Drosophila cell (S2) lines that produce a truncated form of fasciclin I.
The soluble fasciclin I expressed in S2 cells contains significantly less carbohydrate (~15 kDa) as compared to the molecules expressed in CHO cells. Therefore, S2-derived
fasciclin I may be more suitable for crystallization. Biochemical characterization of the expressed fasciclin I indicates that fasciclin I exists as a monomer in solution, an observation consistent with homophilic interaction properties only if the interaction is of low affinity. Electron micrographs of fasciclin I suggest that it has a compact rectangular shape with no obvious flexible linker regions, in contrast to what has been seen in electron
microscopic studies of other adhesion molecules. Circular dichroism analysis suggests that fasciclin I contains significant amounts of α-helical structure, which together with the electron microscopic results, suggests that its structure is substantially different from the β-sheet structures predicted for adhesion molecules that are members of the immunoglobulin superfamily and/or contain fibronectin III repeats. Future structural and functional studies of fasciclin I will ultimately increase our understanding of neuronal cell surface recognition
and axon guidance.